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Dataset Information
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Dataset Name:
Multiplexed imaging of protein levels and protein phosphorylation states in the MCF 10A breast cell line treated with nine kinase inhibitors

Dataset Description:
The MCF 10A breast cell line was plated in 96-well plates and treated with different doses of one of 9 kinase inhibitors (Getifinib, Erlotinib, Lapatinib, PD0325901, Selumetinib, MK2206, Triciribine, Dactolisib, and Torkinib) for 24 or 48 hr before being fixed and analyzed by immunofluorescence microscopy according to the protocol described below.

--Data in Package:
20266.txt

--Metadata in Package:
Small_Molecule_Metadata.txt
Protein_Metadata.txt
Antibody_Metadata.txt
Cell_Line_Metadata.txt

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20266

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20266/

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Assay Information
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Assay Protocol:
1. MCF 10A cells were plated at 5,000 cells per well in duplicate 96-well plates and grown for 24 hr. Cell growth conditions are reported in PMID: <a href = "https://www.ncbi.nlm.nih.gov/pubmed/?term=27135972" target = "_blank">27135972</a>. Cells were grown under the reported conditions for 24 hr and then treated with the indicated dose of Gefitinib, Erlotinib, Lapatinib, PD0325901, Selumetinib, MK2206, Triciribine, Dactolisib, or Torkinib for 24 or 48 hrs.<br />
2. After treatment, cells in each well were fixed with 40 uL 2% PFA at room temperature for 10 min and then washed 3 times with 200 uL PBS per wash.<br />
3. To permeabilize the cells, 40 uL ice-cold methanol were added, and the plates were left at room temperature for 10 minutes. The cells were then washed 3 times with 200 uL PBS per wash.<br />
4. The permeabilized cells were blocked by incubation with 50 uL Odyssey blocking buffer at room temperature for 1 hr.<br />
5. Primary antibodies were diluted in 50 uL Odyssey buffer and added to the cells. The cells were incubated overnight at 4C and then washed gently 3 times with 200 uL PBS per wash. Diluted secondary antibodies were added to the cells, and the cells were incubated for 1 hr at room temperature and washed as just described.<br />
6. Hoechst 33342 (1 mg/mL stock) was diluted 1:5000 in 150 uL PBS and incubated with the cells for 15 minutes at room temperature, and the cells were washed gently 3 times with 200 uL PBS.<br />
7. Whole cell blue dye was diluted (1:1000) in 50 uL PBS and incubated with the cells for 15 minutes at room temperature, and the cells again were washed gently 3 times with 200 uL PBS.<br />
8. To image the fluorescent signals, an Operetta microscope (Perkin Elmer) was used with a 10x objective lens and filter setting for 3-channel immunofluorescence (Hoechst, 568 nm, and 647 nm).<br />
9. Image segmentation and analysis were performed using the Columbus software package. Nuclear segmentation was based on Hoechst 33342 staining, and cell boundaries were segmented based on whole cell blue dye staining.<br />
10. The complete set of images is viewable online through our OMERO server <a href = "http://lincs-omero.hms.harvard.edu/webclient/?show=screen-2301" target = "_blank">lincs-omero.hms.harvard.edu/webclient/?show=screen-2301</a>. Lookup tables for antibody information and for data file data column definitions also are contained within the downloadable data package for this dataset.<br />

Date Updated:
2016-09-29

Date Retrieved from Center:
11/13/2015

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Metadata Information
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Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
[/generic/reagents_studied]
